Clinical Features of Autoimmune Autonomic Ganglionopathy and the Detection of Subunit-Specific Autoantibodies to the Ganglionic Acetylcholine Receptor in Japanese Patients

Autoimmune autonomic ganglionopathy (AAG) is a rare acquired channelopathy that is characterized by pandysautonomia, in which autoantibodies to ganglionic nicotinic acetylcholine receptors (gAChR) may play a central role. Radioimmunoprecipitation (RIP) assays have been used for the sensitive detection of autoantibodies to gAChR in the serum of patients with AAG. Here, we developed luciferase immunoprecipitation systems (LIPS) to diagnose AAG based on IgGs to both the α3 and β4 gAChR subunits in patient serum. We reviewed the serological and clinical data of 50 Japanese patients who were diagnosed with AAG. With the LIPS testing, we detected anti-α3 and -β4 gAChR antibodies in 48% (24/50) of the patients. A gradual mode of onset was more common in the seropositive group than in the seronegative group. Patients with AAG frequently have orthostatic hypotension and upper and lower gastrointestinal tract symptoms, with or without anti-gAChR. The occurrence of autonomic symptoms was not significantly different between the seropositive and seronegative group, with the exception of achalasia in three patients from the seropositive group. In addition, we found a significant overrepresentation of autoimmune diseases in the seropositive group and endocrinological abnormalities as an occasional complication of AAG. Our results demonstrated that the LIPS assay was a useful novel tool for detecting autoantibodies against gAChR in patients with AAG

Autonomic function tests at baseline of anti-gAChR Ab positive AAG patients.

<p>Anti-gAChRα3 Ab = ganglionic acetylcholine receptor α3 antibody; Anti-gAChRβ4 Ab = ganglionic acetylcholine receptor β4 antibody; A.I. = Antibody Index; OH = orthostatic hypotension; HUTT = head-up tilt test; CV R-R = CV = coefficient of variation R-R interval; H/M ratio = heart-to-mediastinum ratio in ¹²³I-MIBG myocardial scintigraphy; TST = thermoregulatory sweat test; NE = norepinephrine; SSR = sympathetic skin response; QSART = quantitative sudomotor axon reflex test; CSF = cerebrospinal fluid</p><p>Autonomic function tests at baseline of anti-gAChR Ab positive AAG patients.</p

LIPS for gAChR in the sera from patients with autoimmune autonomic ganglionopathy (AAG) and controls.

<p>We tested the sera from patients with AAG, disease controls (DC), and healthy controls (HC). a) Anti-gAChRα3 antibodies were detected in 23 samples. The mean anti-gAChRα3 antibody level in the HC was 0.305 antibody index (A.I.), which was significantly lower than in the AAG samples with a mean level of 1.210 A.I. (<i>p</i> < 0.001). b) Anti-gAChRβ4 antibodies were also detected in seven samples, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118312#pone.0118312.g004" target="_blank">Fig. 4B</a> (<i>p</i> = 0.005). The mean anti-gAChRβ4 antibody level in the HC was 0.367 A.I., which was significantly lower than the measn level of 0.618 A.I. in the AAG samples (<i>p</i> < 0.001).</p

Schematic representation of the acetylcholine receptor (AChR) α3-<i>Gaussia</i> luciferase (GL) ⁸⁹⁹⁰.

<p>For the ganglionic AChR (gAChR)-LIPS assay, human embryonic kidney (HEK) 293 cells were transfected with an expression plasmid for the gAChRα3 or β4-GL reporter.</p

Clinical features of patients with AAG/APD.

<p>a. We reviewed the 26 male patients only.</p><p>b. Numbness, mental symptom, dementia, character change, and back pain</p><p>c. Amenorrhea, eating disorder, SIADH (Syndrome of inappropriate secretion of antidiuretic hormone), and panhypopituitarism</p><p>d. Still disease, PBC (primary biliary cirrhosis), Hashimoto disease, PMR (polymyalgia rheumatica), SLE (systemic lupus erythematosus), SS (Sjögren's syndrome), Graves’ disease, RA (rheumatoid arthritis), fibromyalgia, and other autoantibodies positive</p><p>e. Ovarian tumor, pancreas cancer, mediastinal tumor, and paranasal cancer</p><p>Clinical features of patients with AAG/APD.</p

The Luciferase Immunoprecipitation Systems (LIPS).

<p>The soluble fractionated component from the solubilized HEK 293F cells, including the gAChRα3 or β4-GL, reacted with human serum, and the specific luciferase activities of the gAChRα3 or β4-GL were found with the luminometer. The <i>in vitro</i> LIPS assay can quantitatively evaluate an interaction between an antigen and an antibody with high sensitivity and without a radioisotope.</p

Confirmation of the LIPS assay system for the gAChRα3 or β4 with ready-made antibodies.

<p>The anti-gAChRα3 antibody (H-100) and the anti gAChRβ4 antibody (S-15) bound the gAChRα3-GL and the gAChRβ4 reporters, respectively in a dose-dependent manner (a and b). The X-axis indicates the amount of ready-made gAChRα3 or β4 antibody used. The Y-axis indicates the gAChRα3 or β4-GL activity. The line with closed diamonds shows the results that were obtained in this experiment.</p

Clinical and autonomic characteristics at baseline of anti-gAChR Ab positive AAG patients.

<p>Initial symptoms were expressed in bold.</p><p>AE = antecedent event; OH = orthostatic hypotension; OI = orthostatic intolerance; HI = heat intolerance; AH = anhidrosis; GI = gastrointestinal tract symptoms; α3 Ab = ganglionic acetylcholine receptor α3 antibody; β4 Ab = ganglionic acetylcholine receptor β4 antibody; A.I. = Antibody Index; AE = antecedent event; OI = orthostatic intolerance; OH = orthostatic hypotension; HI = heat intolerance; AH = anhidrosis; GI = gastrointestinal tract symptoms; Dept = department</p><p>a. Subacute = peak of autonomic failure within 3 months; gradual = gradual onset of chronic autoimmune autonomic ganglionopathy with the peak of autonomic failure after 3 months.</p><p>b. Patient 2 = fever up; 7 = epididymitis; 21 = influenza virus type A infection; 23 = fever up and cough.</p><p>c. Patient 4 and 7 = Adie’s tonic pupil. The other cases had the abnormality of papillary reflex to light bilaterally or unilaterally.</p><p>d. Patient 1 = constipation; 2 = constipation; 3 = early satiety and vomiting; 4 = constipation; 5 = constipation; 6 = constipation, ileus, and sigmoid volvulus suspected; 7 = early satiety, vomiting, alternate stool abnormality, abdominal pain, and taste impairment; 8 = constipation; 9 = constipation; 10 = constipation; 11 = anorexia and diarrhea; 12 = diarrhea and achalasia; 13 = constipation, early satiety, vomiting, and ileus; 14 = abdominal pain, anorexia, and diarrhea; 15 = diarrhea; 17 = constipation, anorexia, and achalasia; 18 = alternate stool abnormality, abdominal pain, and anorexia; 20 = constipation; 21 = constipation; 22 = early satiety, ileus, alternate stool abnormality, and abdominal pain; 23 = constipation, vomiting, and achalasia; 24 = constipation.</p><p>Clinical and autonomic characteristics at baseline of anti-gAChR Ab positive AAG patients.</p

Comparative Analyses of Copy-Number Variation in Autism Spectrum Disorder and Schizophrenia Reveal Etiological Overlap and Biological Insights

Summary: Compelling evidence in Caucasian populations suggests a role for copy-number variations (CNVs) in autism spectrum disorder (ASD) and schizophrenia (SCZ). We analyzed 1,108 ASD cases, 2,458 SCZ cases, and 2,095 controls in a Japanese population and confirmed an increased burden of rare exonic CNVs in both disorders. Clinically significant (or pathogenic) CNVs, including those at 29 loci common to both disorders, were found in about 8% of ASD and SCZ cases, which was significantly higher than in controls. Phenotypic analysis revealed an association between clinically significant CNVs and intellectual disability. Gene set analysis showed significant overlap of biological pathways in both disorders including oxidative stress response, lipid metabolism/modification, and genomic integrity. Finally, based on bioinformatics analysis, we identified multiple disease-relevant genes in eight well-known ASD/SCZ-associated CNV loci (e.g., 22q11.2, 3q29). Our findings suggest an etiological overlap of ASD and SCZ and provide biological insights into these disorders. : Kushima et al. perform comparative analyses of CNVs in ASD and SCZ in a Japanese population. They identify pathogenic CNVs and biological pathways in each disorder with significant overlap. Patients with pathogenic CNVs have a higher prevalence of intellectual disability. Disease-relevant genes are detected in eight well-known ASD/SCZ-associated CNV loci. Keywords: autism spectrum disorder, schizophrenia, copy-number variation, array comparative genomic hybridization, genetic overlap, Japanese population, oxidative stress response, genome integrity, lipid metabolism, gene ontolog